Isolation and characterization of a potentially novel Siphoviridae phage (vB_SsapS-104) with lytic activity against Staphylococcus saprophyticus isolated from urinary tract infection.

Antibiotic resistance is increasing among Staphylococcus saprophyticus strains isolated from urinary tract infection. This necessitates alternative therapies. For this, a lytic phage (vB_SsapS-104) against S. saprophyticus, which formed round and clear plaques on bacterial culture plates, was isolated from hospital wastewater and characterized. Microscopy analysis showed that it had a small head (about 50 nm), tail (about 80 nm), and a collar (about 22 nm in length and 19 nm in width) indicating to be a phage within Siphoviridae family. Phage vB_SsapS-104 showed a large latency period of about 40 min, rapid adsorption rate that was significantly enhanced by MgCl2 and CaCl2, and high stability to a wide range of temperatures and pH values. Restriction analyses demonstrated that phage consists of a double-stranded DNA with an approximate genome size of 40 Kb. BLAST results did not show high similarity (megablast) with other previously identified phages. But, in Blastn, similarity with Staphylococcus phages was observed. Phage vB_SsapS-104 represented high anti-bacterial activity against S. saprophyticus isolates in vitro as it was able to lyse 8 of the 9 clinical isolates (%88.8) obtained from a hospital in Gorgan, Iran. It was a S. saprophyticus-specific phage because no lytic activity was observed on some other pathogenic bacteria tested. Therefore, phage vB_SsapS-104 can be considered as a specific virulent phage against of S. saprophyitcus isolated from urinary tract infection. This study provided the partial genomic characterization of S. saprophyticus phage and its application against urinary tract infection associated with S. saprophyticus. This phage also can be considered as a good candidate for a therapeutic alternative in the future.

Therapeutic effects of bacteriophages against Salmonella gallinarum infection in chickens.

In this study the isolation and characterization of three bacteriophages (ST4, L13, and SG3) infecting Salmonella gallinarum were carried out. They were further tested for their in vivo efficacy in phage therapy. All three phages belong to the Siphoviridae family with isometric heads and non-contractile tails. They have a broad host range among serovars of Salmonella enterica. The burst sizes were observed to be 1670, 80, and 28 for ST4, L13, and SG3, respectively. The in vivo efficacy of the phages was tested in chickens. Layer chickens were challenged with S. gallinarum, whereas contact chickens were cohabited without direct challenge. Each bacteriophage was orally inoculated in the form of feed additives. Mortality was observed and S. gallinarum was periodically re-isolated from the livers, spleens, and cecums of the chickens. Bacterial re-isolation from the organs and mortality decreased significantly in both challenged and contact chickens treated with the bacteriophages compared with untreated chickens serving as the control. The three bacteriophages may be effective alternatives to antibiotics for the control of fowl typhoid disease in chickens.

Safety analysis of a Russian phage cocktail: from metagenomic analysis to oral application in healthy human subjects.

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

Characterization of phages virulent for Robinia pseudoacacia Rhizobia.

Three lytic phages (PhiRP1, PhiRP2, and PhiRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100-200 phage particles per one infected cell) and rise periods (90-120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4-93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.